The ␤ 2 integrins are important for transendothelial migration of leukocytes as well as for T-cell activation during antigen presentation. Despite abundant expression of ␤ 2 integrins on antigen-presenting cells (APCs), their functional relevance for antigen presentation is completely unclear. We show here that dendritic cells (DCs) from CD18-deficient mice, which lack all functional ␤ 2 integrins, have no defect in antigen presentation. Moreover, DCs from normal mice express inactive ␤ 2 integrins that do not become activated on contact with T cells, at least in vitro. Pharmacologic activation of ␤ 2 integrins on DCs results in a significant reduction of their T cell-activating capacity. This effect is mediated by Mac-1 (CD11b/CD18) on DCs because it could be reversed via blocking antibodies against CD18 and CD11b. Furthermore, the antigen-presenting capacity of macrophages, which express constitutively active ␤ 2 integrins, is significantly enhanced on Mac-1 blockade. We therefore conclude that active CD11b/ CD18 ( IntroductionThe ␤ 2 integrins (CD11/CD18) are heterodimeric leukocyte adhesion molecules exclusively expressed on hematopoietic cells. They play an important role for cell-to-cell contacts between leukocytes as well as for contacts between leukocytes and endothelial cells. 1,2 The common ␤ chain (CD18) associates with 4 different ␣ subunits, ␣L, ␣M, ␣X, and ␣D, forming distinct functional heterodimers termed leukocyte functional antigen-1 (LFA-1, CD11a/CD18), Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18), and CD11d/CD18. [2][3][4] These interact with more than 20 ligands, many of which belong to the family of ICAMs. 5,6 Adhesion between T cells and antigen-presenting cells (APCs) is necessary for the formation of the immunologic synapse (IS). 7,8 Interactions between LFA-1 on the surface of T cells and intercellular adhesion molecules ICAM-1, 2, and 3 (in humans) on the APC surface were described to be involved in formation of the IS. 9,10 Moreover, ligation of LFA-1 on T cells is required for optimal activation and differentiation of T cells, [11][12][13][14][15][16][17][18][19][20][21] although the downstream signaling events are still not fully understood. On T cells, experiments with blocking antibodies against LFA-1 and with LFA-1-deficient T cells have shown that those T cells are impaired in numerous effector functions. [22][23][24] Dendritic cells (DCs), on the other hand, are known to be the most relevant APCs in the immune system. 25,26 DCs express at least 3 ␤ 2 integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/ CD18), and gp150,95 (CD11c/CD18). 27,28 Compared to LFA-1 on T cells, very little information is available about the role and function of ␤ 2 integrins on DCs, especially regarding the process of antigen presentation and T-cell activation. Studies with splenocytes of mice deficient in CD11a, CD11b, CD11c, and CD11d showed impaired T-cell activation in CD11a-, CD11b-, and CD11d-deficient splenocytes on stimulation with superantigen, but this was due to dysfunctional T cells rather than a de...

Toll-like receptor (TLR) ligands lead to the induction of proinflammatory cytokines and are potent enhancers of specific immune responses. We show here that a single systemic dose of R-848, a ligand for TLR7, potently enhanced hapten sensitization during the induction of contact hypersensitivity (CHS). However, R-848 administration also resulted in a rapid and almost complete depletion of leukocytes from the blood. This effect was transient and was associated with general induction of endothelial adhesiveness. In response to R-848, endothelial cells up-regulated adhesion molecules in vitro and in vivo and leukocytes exhibited increased rolling on endothelia in R-848-treated animals. Adhesion molecule induction appeared to be a direct effect, because endothelial cells expressed TLR7 in vitro and in vivo. After R-848 treatment, the tissue residence time of leukocytes was markedly prolonged in all major peripheral organs. The resulting transiently reduced availability of peripheral-blood leukocytes (PBLs) ( IntroductionToll-like receptors (TLRs) are a family of mammalian proteins expressed on a variety of cell types of the immune system. 1 TLRs are able to recognize specific patterns conserved in microorganisms. TLR triggering leads to the induction of inflammatory responses and induces the development of specific immunity. 1 Consequently, specific TLR ligands, such as polyinosinicpolycytidylic acid (poly I:C), lipopolysaccharide (LPS), imidazoquinolines, or CpG oligodeoxynucleotides, have been employed as powerful immune adjuvants and may enhance specific antitumor and antiviral immunity. 2,3 TLR7 is predominantly expressed by plasmacytoid dendritic cells (DCs), myeloid DCs, 4 and B cells 5 and recognizes ssRNA. 6,7 R-848 and the structurally related compound imiquimod (the active substance of the drug Aldara; 3M Pharmaceuticals, St Paul, MN) are artificial ligands for TLR7. 8,9 These imidazoquinolines are immune response modifiers that possess potent antiviral 10 and antitumoral activity when applied topically to the skin. 11 This activity is in part mediated by the induction of type I interferons and inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␣ (IL-1␣). 12 Plasmacytoid DCs have been found to be major targets of R-848, producing large amounts of interferon-␣ and IL-12p40 as well as up-regulating activation markers in response to R-848 both in vitro 13,14 and in vivo. 14 In addition, topical imiquimod has been shown to induce emigration of resident LCs from murine skin, thereby amplifying allergic contact hypersensitivity (CHS) reactions 15 as well as inducing the migration of immature human DCs into draining lymph nodes of cancer patients. 16 Imidazoquinolines also strongly enhance T helper-1 (Th1)-type immune responses: They induce IL-12 in human Langerhans cells (LCs) 17 and lead to production of high amounts of IFN-␥ and to suppression of IL-4 and IL-5 production by T cells in cultures of human peripheral-blood leukocytes (PBLs) and murine spleen cells. 18 Interestingly, pe...

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10+/+, IL-10+/−, and IL-10−/− mice were chronically irradiated with UV. IL-10+/+ and IL-10+/− mice developed skin cancer to similar extents, whereas IL-10−/− mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10−/− mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10−/− mice showed impaired suppressor function when cocultured with conventional CD4+CD25− T cells. CD4+CD25− T cells from IL-10−/− mice produced increased amounts of IFN-γ and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10−/− mice, suggesting strong Th1-drived immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10−/− mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10−/− animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.

Recently, it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study, we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules, thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines, particularly IL-12, whereas the expression of costimulatory molecules remained unchanged. Consequently, synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore, we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However, noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally, we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment, suggesting an autocrine action of type I IFNs.

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Regulatory T cells are promising candidates for the modulation of inflammation and autoimmunity. To generate regulatory T cells in vitro, we have infected naïve CD4 + CD25À T cells with a retrovirus encoding the transcription factor Foxp3. Foxp3-infected T cells are similar to naturally occurring regulatory T cells as evidenced by surface marker expression and function. To investigate the effects of Foxp3-infected T cells on contact hypersensitivity (CHS) responses, sensitized mice were injected with Foxp3-or control virusinfected T cells. Only injection of Foxp3-infected T cells into sensitized mice significantly inhibited CHS compared to controls, indicating that Foxp3-infected T cells are suppressive in vivo. These findings prompted treatment of autoimmune-prone CD40L transgenic (tg) mice, which develop a severe systemic autoimmune disease including autoreactive T cells and autoantibodies, with Foxp3-infected T cells. Interestingly, injections of Foxp3-infected T cells into CD40L tg mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8 + T cells. Strikingly, treatment with Foxp3-infected T cells reduced serum concentrations of antinuclear antibodies in CD40L tg mice, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function. Together, these findings indicate that newly in vitro-generated regulatory T cells can be successfully used to treat inflammatory and ongoing autoimmune disorders.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b−/− DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b−/− DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC–T cell contact dynamics and T cell polarization. Accordingly, Myo9b−/− mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b−/− DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil–forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.